US5585474A - DNA encoding protein possessing metastasis-inhibitory activity - Google Patents

DNA encoding protein possessing metastasis-inhibitory activity Download PDF

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US5585474A
US5585474A US08/555,860 US55586095A US5585474A US 5585474 A US5585474 A US 5585474A US 55586095 A US55586095 A US 55586095A US 5585474 A US5585474 A US 5585474A
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protein
seq
metastasis
amino acid
inhibitory activity
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Kanso Iwaki
Tsunetaka Ohta
Masahi Kurimoto
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Hayashibara Seibutsu Kagaku Kenkyujo KK
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Hayashibara Seibutsu Kagaku Kenkyujo KK
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells
    • C12N5/0694—Cells of blood, e.g. leukemia cells, myeloma cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/70—Undefined extracts
    • C12N2500/72—Undefined extracts from bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/05—Adjuvants
    • C12N2501/052—Lipopolysaccharides [LPS]

Definitions

  • the present invention relates to a novel protein, and a DNA coding said protein, as well as to the preparation of said protein.
  • cancers are mainly attained by surgical operations, chemotherapies and radiotherapies.
  • chemotherapies and radiotherapies Although most of cancers may be cured by such a treatment, a part of viable cancer cells remaining after such a treatment may be scattered throughout the body of a cancer patient, and may cause a more serious cancer-metastasis and even shorten the patient's life span. If the metastasis of cancers can be inhibited, cancer patients's pain would be relieved, and their life spans would be extended much more. Therefore, the development of agents which effectively inhibit the metastasis of cancers has been in a great demand. In general, the metastasis of cancers, however, has been considered to occur via a complicated process, and this hinders the realization of satisfiable cancer metastasis-inhibitory agents.
  • One object of the present invention is to provide a novel protein which effectively inhibits the metastasis of cancers.
  • Another object of the invention is to provide a DNA coding said protein.
  • the present inventors studied substances which inhibit the metastasis of cancers.
  • the present inventors continued studies in order to obtain a novel cancer metastasis-inhibitory substance, and, as a result eventually found cancer metastasis-inhibitory activity in a culture supernatant of HPB-MLT cell (FERM BP-2430), an established cell line derived from human T-cell leukemia, which had been stimulated in a nutrient culture medium with BCG and LPS.
  • the present inventors revealed that the entity of the activity is a protein having the following physicochemical properties:
  • Soluble in water, physiological saline and phosphate buffer Soluble in water, physiological saline and phosphate buffer
  • the present inventors isolated a protein possessing cancer metastasis-inhibitory activity from a culture of HPB-MLT cell (FERM BP-2430) stimulated with BCG and LPS.
  • the present inventors revealed the physicochemical properties of said protein and found that it has the amino acid sequence as shown in Chemical formula 1.
  • SEQ ID NO: 4 The wording "substantially has the amino acid sequence as shown in Chemical formula 1" as referred to in the invention means that it should not be restricted to that as shown in Chemical formula 1 and shall include all the homologous variants thereof.
  • the present invention includes any protein having a partial amino acid sequence in the amino acid sequence as shown in Chemical formula 1 as long as it possesses the same physicochemical properties as the above-mentioned protein. ##STR1##
  • the present inventors screened DNAs which might code the present protein from HPB-MLT cell, and found that the present protein contained the base sequence as shown in Chemical formula 2(SEQ ID NO: 3).
  • the DNA according to the present invention is not restricted to that as shown in Chemical formula 2.
  • the wording "substantially has the base sequence as shown in Chemical formula 2" as referred to in the invention means that it has the whole or a part of the base sequence of Chemical formula 2.
  • the base sequences usable in the invention are, for example, those formed by a genetic code degeneracy wherein one or more bases in Chemical formula 2 are replaced with other bases, those which code the aforesaid homologous variants, and those which are complemental to the base sequence as shown in Chemical formula 2.
  • the complementary base sequences may be wholly or partially complemental to that as shown in Chemical formula 2. ##STR2##
  • the present invention provides a process to prepare the above-mentioned protein, comprising culturing a cell "capable of producing said protein in a nutrient culture medium to form said protein, and recovering the resultant protein from the culture.
  • a cell are established cell lines derived from human T-cell leukemia such as HPB-MLT cell and MOLT-4 cell (ATCC CRL 1582), and not restricted to thcse of human origin.
  • cells of human origin cells of animal origin and microorganisms can be advantageously used in the invention as long as they can inherently produce the present protein, and those which had been introduced with the present DNA by conventional cell fusion or genetic engineering technique can be advantageously used in the invention as long as the present protein is recovered from their cultures.
  • the cells usable in the present process are not restricted to those described in the present specification, and, if necessary the present protein can be prepared by culturing any one of the cells in a nutrient culture medium while stimulating it with an adequate stimulant such as BCC and LPS, and recovering the resultant protein having a metastasis-inhibitory activity from the resultant cells or supernatant.
  • the cultivation of such a cell is carried out according to conventional techniques for animal cells and microorganisms.
  • Conventional nutrient culture media containing vitamins, minerals, carbohydrates and the like can be employed in the invention.
  • the recovering methods suitably used in the invention are two or more methods usually used in the purification of physiologically-active proteinous substances: For example, salting out, dialysis, centrifugation, gel filtration chromatography, ion-exchange chromatography, affinity chromatography, electrophoresis, isoelectrofocusing and isoelectric fractionation are suitably used in combination.
  • the present invention attains the aforesaid object, and encompasses a novel protein possessing a metastasis-inhibitory activity, a DNA coding said protein, and the preparation of said protein.
  • mice 5 or more BALB/c nude mice were injected through their tail veins with 0.2ml of phosphate buffer containing a test specimen 3 times in total before the cell transplantation, i.e. at 2 day, 1 day and 3 hours before the cell transplantation. From the next day of the transplantation of 2 ⁇ 10 6 RPMI 4788 cells per mouse, the mice were successively injected similarly as above with a test specimen one shot per day over a period of 7 days. In a control group, mice were similarly treated as in the test group except for using phosphate buffer free of the test specimen. On the 21st day after the cell transplantation, nude mice were sacrificed, and the number of metastatic nodules formed on the surfaces of the lungs was macroscopically counted.
  • test specimen had a positive activity when the following requirements were fulfilled: (i) The mean value of numbers of lung metastatic nodules formed in the mice in the control group was 50 or more; (ii) the mean value of those in the test group was lowered to 1/2 or lower against that in the control group; and (iii) the reduction level in (ii) was evaluated as statistically significant.
  • a new born hamster was first injected with a serum of anti-hamster thymus prepared from rabbits in usual manner, then subcutaneously transplanted with HPB-MLT cells, and bred for 4 weeks in usual manner.
  • About 20 g weight tumor subcutaneously formed in the hamster was cut into pieces, dispersed, washed with serum-free RPMI 1640 medium, and suspended in a fresh preparation of the same medium to give a concentration of 5 ⁇ 10 6 cells/ml.
  • the cell suspension was added with 10 ⁇ g/ml BCG and incubated at 37° C. for one day. Thereafter, the resultant cell suspension was added with one ⁇ g/ml LPS, incubated for one day, and centrifuged to obtain a supernatant.
  • the column was fed with "Polybuffer® 74 (pH 4.0)", commercialized by Pharmacia LKB, Uppsala, Sweden, to fractionate the supernatant, and the resultant fractions were respectively dialyzed against phosphate-buffered saline (PBS), followed by assaying each fraction for cancer metastasis-inhibitory activity. As a result, it was revealed that the fractions eluted between a pH range of 5.0-6.5 had cancer metastasis-inhibitory activity.
  • the active fractions were pooled, and refractionated on a column packed with "Sephacryl S-200", a product commercialized by Pharmacia LKB Uppsala, Sweden.
  • the resultant fractions were assayed for their cancer metastasis-inhibitory activity to test whether or not that the fractions, eluted with a ratio of (Elution volume)/(Void volume) in the range of 0.5-0.65, might have the activity.
  • the active fractions were pooled and dialyzed against 10 mM potassium phosphate buffer (pH 7.4).
  • the solution in a dialytic bag was fed to a column packed with "DEAE-SPW", a product of Tosoh Corporation, Tokyo, Japan, preequilibrated with 10 mM potassium phosphate buffer (pH 7.4), and eluted with a liner gradient of 10-500 mM potassium phosphate buffer (pH 7.4).
  • a substance with cancer metastasis-inhibitory activity was eluted in fractions with about 70 mM potassium phosphate.
  • the fractions thus obtained were pooled and dialyzed against 10 mM sodium phosphate buffer (pH 6.8), and fed to a hydroxyapatite column commercialized by Toa Nenryo Kogyo K.K., Tokyo, Japan, preequilibrated with 10 mM sodium phosphate buffer (pH 6.8).
  • cancer metastasis-inhibitory activity was found in non-adsorbed fractions which were then fed to a column packed with "Mono-P", a product of Pharmacia LKB, Upssala, Sweden, preequilibrated with 25 mM bis-tris-iminodiacetate buffer (pH 7.1), and eluted with "Polybuffer® 74 (pH 4.0)", a product of Pharmacia LKB, Uppsala, Sweden, followed by isolating the present protein with cancer metastasis-inhibitory activity.
  • “Mono-P” a product of Pharmacia LKB, Upssala, Sweden
  • Polybuffer® 74 pH a product of Pharmacia LKB, Uppsala, Sweden
  • the protein was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (abbreviated as "SDS-PAGE” hereinafter), and the molecular weight was determined to be 45,000 ⁇ 5,000 based on the relative mobility of the protein against marker proteins.
  • SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis
  • the isoelectric point of the protein was estimated to be 5.7 ⁇ 0.5 based on the pHs of eluates on a column chromatography using Mono-P column;
  • the protein was subjected to SDS-PAGE, and a band corresponding to the molecular weight of about 45,000 in the resultant gel was isolated by cutting.
  • the resultant gel piece was soaked in 100 mM Tris-HCl buffer (pH 9.0) containing 0.1% sodium dodecyl sulfate (SDS) at 37° C. for one hour, and digested at 37° C. overnight by the addition of 5 ⁇ g/ml lysyl endopeptidase, an enzyme specimen commercialized by Wako Pure Chemical Industries, Ltd., Tokyo, Japan.
  • the supernatant obtained from the resultant was fractionated on a reverse-phase chromatography using a column of "C-18", commercialized by VYDAC, Hesperia, USA, equipped with a precolumn of DEAE, followed by recovering peptide fragments which were then analyzed on "470A", an amino acid sequencer commercialized by Applied Biosystems Inc., Foster City, USA.
  • the results were as shown in Chemical formulas 3, 4, 5 and 6.
  • the protein was soluble in water, physiological saline and phosphate buffer.
  • the protein was tested for cancer metastasis-inhibitory activity with the method in Experiment 1. As a result, the number of metastatic nodules was 314 ⁇ 169 in a control group consisting of 5 nude mice, while that in a test group, wherein 5 nude mice were administered with a solution containing 250 ⁇ g/ml of the protein, was 100 ⁇ 43. These confirmed that the protein exhibited a strong cancer metastasis-inhibitory activity.
  • the protein was dissolved in phosphate buffer (pH 7.2) and allowed to stand at 100° C. for 30 minutes, followed by determining the residual activity with the method in Experiment 1 to give no activity. Thus, it was revealed that the protein was inactivated under the conditions.
  • mice of the present protein was tested for acute toxicity.
  • the LD50 in mice of the protein was 50 mg/kg or higher when intravenously administered to the mice, and this revealed that the toxicity was extremely low.
  • HPB-MLT cells obtained by the method in Experiment 2 were suspended in a serum-free RPMI 1640 medium to give a concentration of 5 ⁇ 10 6 cells/ml.
  • the cell suspension was added with 10 ⁇ g/ml BCG and incubated at 37° C. for one day.
  • the culture thus obtained was added with one g/ml LPS and incubated for 4.5 hours.
  • the resultant culture was centrifuged to obtain cells which were then solubilized with 4M guanidium isocyanate and homogenized.
  • the resultant homogenate was overlaid on 5.7M cesium chloride, and the mixture was centrifuged at 25,000 rpm for 17 hours to obtain the whole RNAs of the cells.
  • Poly (A) + RNA was purified from the whole RNAs on "Oligotex"-dT30 ⁇ Super>", a bead for purification of poly (A) + RNA commercialized by Daiichi Pure Chemicals, Tokyo, Japan.
  • the purified poly (A) + RNA was treated with "cDNA synthesis system plus", a product of Amersham International plc, Buckinghashire, England, to synthesize a cDNA under the direction of the appended manual.
  • the cDNA thus obtained was ligated with a ⁇ gt10 phage DNA by using "cDNA cloning system ⁇ gt10", a product of Amersham International plc, Buckinghashire, Englnad.
  • the resultant recombinant phage DNA was packaged by "Lambda( ⁇ ) in vitro packaging kit", a product of Amersham International plc, Buckinghashire, England, to obtain a cDNA library of HPB-MLT cell.
  • the resultant was admixed with a soft agar, and the resultant mixture was overlaid on a hard-agar plate and solidified.
  • the resultant agar plate containing the microorganisms were incubated at 37° C.
  • Hybond-N filter a membrane filter of Amersham International plc, Buckinghamshire, England, to transfer the phage and to fix the phage DNA on the membrane filter.
  • the membrane filter was soaked in a solution of a salmon sperm DNA commercialized by Sigma Chemical company, St., Louis, USA, to effect prehybridization, followed by screening positive clones by the southern hybridization with the radiolabeled DNA probes in Experiment 5-2.
  • a phage DNA was isolated and digested with a restriction enzyme EcoRI, and the resultant fragments were separated on SDS-PAGE to obtain an inserted DNA fragment of about 1.5 kbp which was then ligated with a pUC18 plasmid with a ligation kit commercialized by Amersham International plc, Buckinghashire, England, to obtain a recombinant plasmid.
  • the recombinant plasmid thus obtained was introduced in usual manner into E. coli to obtain a recombinant microorganism, and from which a plasmid DNA was prepared.
  • the dideoxy chain termination method was applied to the resultant plasmid DNA to reveal the base sequence of the present protein. Based on the base sequence, the amino acid sequence of the present protein was estimated. As a result, it was revealed that the present DNA consisted of 1,224 bases and coded a protein consisting of 408 amino acids. The estimated amino acid sequence encompassed the amino acid sequences as shown in Chemical formulas 3, 4, 5 and 6 in Experiment 3.
  • MOLT-4 cell (ATCC CRL 1582), an established cell line derived from human T-cell leukemia commercialized by Dainippon Pharmaceutical Co., Ltd., Tokyo, Japan, were cultured in a nutrient culture medium while stimulating the cells with BCG and LPS.
  • a supernatant prepared from the resultant culture was treated to obtain the present protein possessing cancer metastasis-inhibitory activity.
  • the yield was about 40 ⁇ g per 50L of the culture supernatant.
  • the protein had the same physicochemical properties as the one in Experiment 3.
  • the protein according to the present invention effectively inhibits the metastasis of cancers, and this renders it advantageously useful as prophylactic-, therapeutic- and diagnostic-agents for cancer metastases.
  • the toxicity of the present protein is satisfactorily low, because of this it can be systematically administered to patients in the form of an injection, sublingual agent, and the like.
  • the present protein having the aforesaid advantages is prepared in an industrial scale by the present preparation.
  • the DNA coding the present protein is useful in the preparation of the present protein by genetic engineering techniques.

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Abstract

Disclosed is a novel protein which has a molecular weight of 45,000±5,000 and pI 5.7±0.5 and exhibits cancer metastasis-inhibitory activity. The protein can be prepared by culturing human cells, animal cells and microorganisms capable of producing the protein in a nutrient culture medium while stimulating them with an inducer such as Bacille Calmette-Gu erin and lipopolysaccharide.

Description

This is a division of parent application Ser. No. 08/127,278 filed Sep. 27, 1993, now U.S. Pat. No. 5,498,697.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a novel protein, and a DNA coding said protein, as well as to the preparation of said protein.
2. Description of the Prior Art
Nowadays, the treatment of cancers is mainly attained by surgical operations, chemotherapies and radiotherapies. Although most of cancers may be cured by such a treatment, a part of viable cancer cells remaining after such a treatment may be scattered throughout the body of a cancer patient, and may cause a more serious cancer-metastasis and even shorten the patient's life span. If the metastasis of cancers can be inhibited, cancer patients's pain would be relieved, and their life spans would be extended much more. Therefore, the development of agents which effectively inhibit the metastasis of cancers has been in a great demand. In general, the metastasis of cancers, however, has been considered to occur via a complicated process, and this hinders the realization of satisfiable cancer metastasis-inhibitory agents.
Although a variety of proteins possessing cancer metastasis-inhibitory activity were reported, the present protein absolutely differs from them. Examples of such a conventional protein are interferons and interleukin 2 which have been reported to have cancer metastasis-inhibitory activity. The present protein clearly differs from such a conventional protein in molecular weight and amino acid sequence. None of conventional proteins have not yet been realized as a cancer metastasis-inhibitory agent. In Japanese Patent Laid-Open No.308,799/90, a cancer metastasis-inhibitory factor produced by cells derived from human hematopoietic tissues is reported and its structure and physicochemical properties are, however, far from substantial elucidation because the description in the patent is vague and it only teaches the molecular weight ranging 10,000-450,000.
OBJECTS OF THE INVENTION
One object of the present invention is to provide a novel protein which effectively inhibits the metastasis of cancers.
Another object of the invention is to provide a DNA coding said protein.
Further object of the invention is to provide the preparation of said protein.
SUMMARY OF THE INVENTION
In order to attain the aforesaid objects, the present inventors studied substances which inhibit the metastasis of cancers. The present inventors continued studies in order to obtain a novel cancer metastasis-inhibitory substance, and, as a result eventually found cancer metastasis-inhibitory activity in a culture supernatant of HPB-MLT cell (FERM BP-2430), an established cell line derived from human T-cell leukemia, which had been stimulated in a nutrient culture medium with BCG and LPS. The present inventors revealed that the entity of the activity is a protein having the following physicochemical properties:
(1) Molecular weight
45,000±5,000;
(2) Isoelectric point
pI=5.7±0.5;
(3) Partial amino acid sequence
Possessing a partial amino acid sequence of Asp-Ser-Glu-Gly-Tyr-Ile-Tyr-Ala-Arg-Gly-Ala-Gln-Asp-Met-Lys (SEQ ID No: 1) or Glu-His-Trp-Ser-His-Asp-Pro-Phe-Glu (SEQ ID NO:2);
(4) Solubility in solvent
Soluble in water, physiological saline and phosphate buffer;
(5) Biological activity
Exhibiting a metastasis-inhibitory activity on RPMI 4788 cell (FERM BP-2429), an established cell line derived from human colon cancer; and
(6) Stability
Inactivated in water at pH 7.2 and 100° C. for 30 minutes.
Stable in water at pH 7.2 and 4° C. for one month.
DETAILED DESCRIPTION OF THE INVENTION
The present inventors isolated a protein possessing cancer metastasis-inhibitory activity from a culture of HPB-MLT cell (FERM BP-2430) stimulated with BCG and LPS. The present inventors revealed the physicochemical properties of said protein and found that it has the amino acid sequence as shown in Chemical formula 1. (SEQ ID NO: 4) The wording "substantially has the amino acid sequence as shown in Chemical formula 1" as referred to in the invention means that it should not be restricted to that as shown in Chemical formula 1 and shall include all the homologous variants thereof. In other words, the present invention includes any protein having a partial amino acid sequence in the amino acid sequence as shown in Chemical formula 1 as long as it possesses the same physicochemical properties as the above-mentioned protein. ##STR1##
Based on the above-mentioned amino acid sequence, the present inventors screened DNAs which might code the present protein from HPB-MLT cell, and found that the present protein contained the base sequence as shown in Chemical formula 2(SEQ ID NO: 3). The DNA according to the present invention is not restricted to that as shown in Chemical formula 2. The wording "substantially has the base sequence as shown in Chemical formula 2" as referred to in the invention means that it has the whole or a part of the base sequence of Chemical formula 2. The base sequences usable in the invention are, for example, those formed by a genetic code degeneracy wherein one or more bases in Chemical formula 2 are replaced with other bases, those which code the aforesaid homologous variants, and those which are complemental to the base sequence as shown in Chemical formula 2. The complementary base sequences may be wholly or partially complemental to that as shown in Chemical formula 2. ##STR2##
Furthermore, the present invention provides a process to prepare the above-mentioned protein, comprising culturing a cell "capable of producing said protein in a nutrient culture medium to form said protein, and recovering the resultant protein from the culture. Examples of such a cell are established cell lines derived from human T-cell leukemia such as HPB-MLT cell and MOLT-4 cell (ATCC CRL 1582), and not restricted to thcse of human origin. Similarly as the cells of human origin, cells of animal origin and microorganisms can be advantageously used in the invention as long as they can inherently produce the present protein, and those which had been introduced with the present DNA by conventional cell fusion or genetic engineering technique can be advantageously used in the invention as long as the present protein is recovered from their cultures.
The cells usable in the present process are not restricted to those described in the present specification, and, if necessary the present protein can be prepared by culturing any one of the cells in a nutrient culture medium while stimulating it with an adequate stimulant such as BCC and LPS, and recovering the resultant protein having a metastasis-inhibitory activity from the resultant cells or supernatant. The cultivation of such a cell is carried out according to conventional techniques for animal cells and microorganisms. Conventional nutrient culture media containing vitamins, minerals, carbohydrates and the like can be employed in the invention. The recovering methods suitably used in the invention are two or more methods usually used in the purification of physiologically-active proteinous substances: For example, salting out, dialysis, centrifugation, gel filtration chromatography, ion-exchange chromatography, affinity chromatography, electrophoresis, isoelectrofocusing and isoelectric fractionation are suitably used in combination.
The present invention attains the aforesaid object, and encompasses a novel protein possessing a metastasis-inhibitory activity, a DNA coding said protein, and the preparation of said protein.
The following experiments will explain the present invention.
EXPERIMENT 1
Assay for metastasis-inhibitory activity
In accordance with the method of Y. Naomoto et al. described in Journal of Cancer Research and Clinical Oncology, Vol.113, pp.544-549 (1987), a metastasis-inhibitory activity was assayed with a model wherein RPMI 4788 cells (FERM BP-2429) are transplanted to nude mice so as to induce lung metastasis.
In a test group, 5 or more BALB/c nude mice were injected through their tail veins with 0.2ml of phosphate buffer containing a test specimen 3 times in total before the cell transplantation, i.e. at 2 day, 1 day and 3 hours before the cell transplantation. From the next day of the transplantation of 2×106 RPMI 4788 cells per mouse, the mice were successively injected similarly as above with a test specimen one shot per day over a period of 7 days. In a control group, mice were similarly treated as in the test group except for using phosphate buffer free of the test specimen. On the 21st day after the cell transplantation, nude mice were sacrificed, and the number of metastatic nodules formed on the surfaces of the lungs was macroscopically counted. It was judged that a test specimen had a positive activity when the following requirements were fulfilled: (i) The mean value of numbers of lung metastatic nodules formed in the mice in the control group was 50 or more; (ii) the mean value of those in the test group was lowered to 1/2 or lower against that in the control group; and (iii) the reduction level in (ii) was evaluated as statistically significant.
EXPERIMENT 2 Preparation of supernatant from culture of HPB-MLT cell stimulated with BCG and LPS
A new born hamster was first injected with a serum of anti-hamster thymus prepared from rabbits in usual manner, then subcutaneously transplanted with HPB-MLT cells, and bred for 4 weeks in usual manner. About 20 g weight tumor subcutaneously formed in the hamster was cut into pieces, dispersed, washed with serum-free RPMI 1640 medium, and suspended in a fresh preparation of the same medium to give a concentration of 5×106 cells/ml. The cell suspension was added with 10 μg/ml BCG and incubated at 37° C. for one day. Thereafter, the resultant cell suspension was added with one μg/ml LPS, incubated for one day, and centrifuged to obtain a supernatant.
EXPERIMENT 3
Purification and physicochemical properties of the present protein
The supernatant in Experiment 2 was concentrated by about 20-fold on "AIL 3013", a membrane module commercialized by Asahi Chemical Ind., Tokyo, Japan, and the concentrate was dialyzed against 25 mM imidazol-HCl buffer (pH 7.4). The resultant solution in a dialytic bag was fed to a column packed with "PBE-94", a product of Pharmacia LKB, Uppsala, Sweden, preequilibrated with 25 mM imidazol-HCl buffer (pH 7.4). The column was fed with "Polybuffer® 74 (pH 4.0)", commercialized by Pharmacia LKB, Uppsala, Sweden, to fractionate the supernatant, and the resultant fractions were respectively dialyzed against phosphate-buffered saline (PBS), followed by assaying each fraction for cancer metastasis-inhibitory activity. As a result, it was revealed that the fractions eluted between a pH range of 5.0-6.5 had cancer metastasis-inhibitory activity. The active fractions were pooled, and refractionated on a column packed with "Sephacryl S-200", a product commercialized by Pharmacia LKB Uppsala, Sweden. The resultant fractions were assayed for their cancer metastasis-inhibitory activity to test whether or not that the fractions, eluted with a ratio of (Elution volume)/(Void volume) in the range of 0.5-0.65, might have the activity. The active fractions were pooled and dialyzed against 10 mM potassium phosphate buffer (pH 7.4). The solution in a dialytic bag was fed to a column packed with "DEAE-SPW", a product of Tosoh Corporation, Tokyo, Japan, preequilibrated with 10 mM potassium phosphate buffer (pH 7.4), and eluted with a liner gradient of 10-500 mM potassium phosphate buffer (pH 7.4). In this case, a substance with cancer metastasis-inhibitory activity was eluted in fractions with about 70 mM potassium phosphate. The fractions thus obtained were pooled and dialyzed against 10 mM sodium phosphate buffer (pH 6.8), and fed to a hydroxyapatite column commercialized by Toa Nenryo Kogyo K.K., Tokyo, Japan, preequilibrated with 10 mM sodium phosphate buffer (pH 6.8). In this case, cancer metastasis-inhibitory activity was found in non-adsorbed fractions which were then fed to a column packed with "Mono-P", a product of Pharmacia LKB, Upssala, Sweden, preequilibrated with 25 mM bis-tris-iminodiacetate buffer (pH 7.1), and eluted with "Polybuffer® 74 (pH 4.0)", a product of Pharmacia LKB, Uppsala, Sweden, followed by isolating the present protein with cancer metastasis-inhibitory activity. Thus, about 70 μg of the present protein was isolated from 50L of the culture supernatant of HPB-MLT cells stimulated with BCG and LPS. The physicochemical properties of the present protein were studied with the isolated protein.
(1) Molecular weight
In accordance with the method of U. K. Laemmli described in Nature, Vol.227, pp.680-685 (1970), the protein was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (abbreviated as "SDS-PAGE" hereinafter), and the molecular weight was determined to be 45,000±5,000 based on the relative mobility of the protein against marker proteins.
(2) Isoelectric point
The isoelectric point of the protein was estimated to be 5.7±0.5 based on the pHs of eluates on a column chromatography using Mono-P column;
(3) Partial amino acid sequence
The protein was subjected to SDS-PAGE, and a band corresponding to the molecular weight of about 45,000 in the resultant gel was isolated by cutting. The resultant gel piece was soaked in 100 mM Tris-HCl buffer (pH 9.0) containing 0.1% sodium dodecyl sulfate (SDS) at 37° C. for one hour, and digested at 37° C. overnight by the addition of 5 μg/ml lysyl endopeptidase, an enzyme specimen commercialized by Wako Pure Chemical Industries, Ltd., Tokyo, Japan. The supernatant obtained from the resultant was fractionated on a reverse-phase chromatography using a column of "C-18", commercialized by VYDAC, Hesperia, USA, equipped with a precolumn of DEAE, followed by recovering peptide fragments which were then analyzed on "470A", an amino acid sequencer commercialized by Applied Biosystems Inc., Foster City, USA. The results were as shown in Chemical formulas 3, 4, 5 and 6.
Chemical formula 3 (SEQ ID NO: 1) Asp-Ser-Glu-Gly-Tyr-Ile-Tyr-Ala-Arg-Gly-Ala-Gln-Asp-Met-Lys
Chemical formula 4 (SEQ ID NO: 2)
Glu-His-Trp-Ser-His-Asp-Pro-Phe-Glu
Chemical formula 5 (SEQ ID NO
Glu-Trp-Gln-Arg-Leu-Gtn-Ser-Asn-Pro-His-Leu-Lys
Chemical formula 6
Leu-Glu-Gly-Gly-Val-Ala-Tyr-Asn-Val-Ile-Pro
(4) Solubility in solvent
The protein was soluble in water, physiological saline and phosphate buffer.
(5) Biological activity
The protein was tested for cancer metastasis-inhibitory activity with the method in Experiment 1. As a result, the number of metastatic nodules was 314±169 in a control group consisting of 5 nude mice, while that in a test group, wherein 5 nude mice were administered with a solution containing 250 μg/ml of the protein, was 100±43. These confirmed that the protein exhibited a strong cancer metastasis-inhibitory activity.
(6) Stability
The protein was dissolved in phosphate buffer (pH 7.2) and allowed to stand at 100° C. for 30 minutes, followed by determining the residual activity with the method in Experiment 1 to give no activity. Thus, it was revealed that the protein was inactivated under the conditions.
While the protein was treated by dissolving it in phosphate buffer (pH 7.2), and allowing the resultant solution to stand at 4° C. for one month, followed by assaying the residual activity similarly as above. As a result, no substantial loss of activity was found, and this revealed that the protein was stable under the conditions.
EXPERIMENT 4
Acute toxicity
By using 7 week-old mice, the present protein was tested for acute toxicity. As a result, the LD50 in mice of the protein was 50 mg/kg or higher when intravenously administered to the mice, and this revealed that the toxicity was extremely low.
Experiment 5
Base sequence coding the present protein
In this experiment, the base sequence of the present protein was determined by conventional method as described by T. Maniatis et al. in Molecular Cloning, A Laboratory Manual, 2nd edition, published by Cold Spring Harbor LaboratoryPress (1989), New York, USA.
Experiment 5-1
Construction of cDNA library of HPB-MLT cell
HPB-MLT cells obtained by the method in Experiment 2 were suspended in a serum-free RPMI 1640 medium to give a concentration of 5×106 cells/ml. The cell suspension was added with 10 μg/ml BCG and incubated at 37° C. for one day. The culture thus obtained was added with one g/ml LPS and incubated for 4.5 hours. The resultant culture was centrifuged to obtain cells which were then solubilized with 4M guanidium isocyanate and homogenized. The resultant homogenate was overlaid on 5.7M cesium chloride, and the mixture was centrifuged at 25,000 rpm for 17 hours to obtain the whole RNAs of the cells. Poly (A)+ RNA was purified from the whole RNAs on "Oligotex"-dT30<Super>", a bead for purification of poly (A)+ RNA commercialized by Daiichi Pure Chemicals, Tokyo, Japan. The purified poly (A)+ RNA was treated with "cDNA synthesis system plus", a product of Amersham International plc, Buckinghashire, England, to synthesize a cDNA under the direction of the appended manual. The cDNA thus obtained was ligated with a λgt10 phage DNA by using "cDNA cloning system λgt10", a product of Amersham International plc, Buckinghashire, Englnad. The resultant recombinant phage DNA was packaged by "Lambda(λ) in vitro packaging kit", a product of Amersham International plc, Buckinghashire, England, to obtain a cDNA library of HPB-MLT cell.
Experiment 5-2
Construction of radiolabeled DNA probe
Based on the partial amino acid sequence as shown in Chemical formula 3 in Experiment 3, base sequences estimable from the amino acid sequence of Glu-Gly-Tyr-Ile-Tyr-Ala (SEQ ID NO: 7) in Chemical formula 3 were synthesized by a DNA synthesizer commercialized by Applied Biosystems, Inc., Foster City, USA. Ninety-six base sequences consisting of 17 synthesized bases are as shown in Table 1.
                                  TABLE 1                                 
__________________________________________________________________________
Probe 1:                                                                  
      GAG GGG TAT ATA                                                     
                     TAT                                                  
                        GC (SEQ ID NO: 8)                                 
      A   A   C   T  C                                                    
          T       C                                                       
          C                                                               
__________________________________________________________________________
As for the partial amino acid sequence as shown in Chemical formula 5, complementary chains of base sequences, estimable from Asn-Pro-His-Leu-Lys (SEQ ID NO:9) in the partial amino acid sequence in Chemical formula 5, were synthesized similarly as above. Ninety-six base sequences consisting of 14 synthesized bases are as shown in Table 2. The DNAs thus obtained were radiolabeled with [γ-32 P]ATP and T4 polynucleotide kinase.
              TABLE 2                                                     
______________________________________                                    
Probe TTG    AGG     TGG   GGG   TT   (SEQ ID NO: 10)                     
2:    A      A       A     A                                              
      T              T                                                    
      C              C                                                    
      TTT    AAG     TGG   GGG   TT                                       
      C      A       A     A                                              
                     T                                                    
                     C                                                    
______________________________________
Experiment 5-3
Screening with radiolabeled DNA probes
A solution of the recombinant λgt10, a cDNA library of HPB-MLT cell prepared in Experiment 5-1, was mixed with an overnight culture of microorganisms of E. coli strain NM 514 in L-broth, and the mixture was incubated at 37° C. for 15 minutes. The resultant was admixed with a soft agar, and the resultant mixture was overlaid on a hard-agar plate and solidified. The resultant agar plate containing the microorganisms were incubated at 37° C. for 8 hours, cooled and overlaid with "Hybond-N filter", a membrane filter of Amersham International plc, Buckinghamshire, England, to transfer the phage and to fix the phage DNA on the membrane filter. In order to prevent a non-specific bonding of the radiolabeled DNA probes with DNAs except for the objective complementary DNA, the membrane filter was soaked in a solution of a salmon sperm DNA commercialized by Sigma Chemical company, St., Louis, USA, to effect prehybridization, followed by screening positive clones by the southern hybridization with the radiolabeled DNA probes in Experiment 5-2. The results in the first screening test with the probe 1 in Table 1 and the second screening test with the probe 2 in Table 2 revealed that 3 positive clones were present among about 600,000 clones. Phage DNAs isolated from the positive clones were digested with a restriction enzyme EcoRI to remove them from vector DNAs, and the length of the inserted fragments were studied on agarose electrophoresis, followed by analyzing a clone having the longest inserted-fragment of about 1.5 kbp.
Experiment 5-4
Base sequence of gene coding the present protein
From the positive clones obtained in Experiment 5-3, a phage DNA was isolated and digested with a restriction enzyme EcoRI, and the resultant fragments were separated on SDS-PAGE to obtain an inserted DNA fragment of about 1.5 kbp which was then ligated with a pUC18 plasmid with a ligation kit commercialized by Amersham International plc, Buckinghashire, England, to obtain a recombinant plasmid. The recombinant plasmid thus obtained was introduced in usual manner into E. coli to obtain a recombinant microorganism, and from which a plasmid DNA was prepared. The dideoxy chain termination method was applied to the resultant plasmid DNA to reveal the base sequence of the present protein. Based on the base sequence, the amino acid sequence of the present protein was estimated. As a result, it was revealed that the present DNA consisted of 1,224 bases and coded a protein consisting of 408 amino acids. The estimated amino acid sequence encompassed the amino acid sequences as shown in Chemical formulas 3, 4, 5 and 6 in Experiment 3.
The following is an example of the preparation of the present protein.
EXAMPLE
Similarly as the method in Experiment 2, MOLT-4 cell (ATCC CRL 1582), an established cell line derived from human T-cell leukemia commercialized by Dainippon Pharmaceutical Co., Ltd., Tokyo, Japan, were cultured in a nutrient culture medium while stimulating the cells with BCG and LPS. Similarly as in Experiment 3, a supernatant prepared from the resultant culture was treated to obtain the present protein possessing cancer metastasis-inhibitory activity. The yield was about 40 μg per 50L of the culture supernatant. The protein had the same physicochemical properties as the one in Experiment 3.
The protein according to the present invention effectively inhibits the metastasis of cancers, and this renders it advantageously useful as prophylactic-, therapeutic- and diagnostic-agents for cancer metastases. The toxicity of the present protein is satisfactorily low, because of this it can be systematically administered to patients in the form of an injection, sublingual agent, and the like.
The present protein having the aforesaid advantages is prepared in an industrial scale by the present preparation.
The DNA coding the present protein is useful in the preparation of the present protein by genetic engineering techniques.
While there has been described what is at present considered to be the preferred embodiments of the invention, it will be understood the various modifications may be made therein, and it is intended to cover the appended claims all such modifications, as fall within the true spirits and scope of the invention.
__________________________________________________________________________
SEQUENCE LISTING                                                          
(1) GENERAL INFORMATION:                                                  
(iii) NUMBER OF SEQUENCES: 11                                             
(2) INFORMATION FOR SEQ ID NO:1:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 15 amino acids                                                
(B) TYPE: amino acid                                                      
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: peptide                                               
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                   
AspSerGluGlyTyrIleTyrAlaArgGlyAlaGlnAspMetLys                             
151015                                                                    
(2) INFORMATION FOR SEQ ID NO:2:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 9 amino acids                                                 
(B) TYPE: amino acid                                                      
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: peptide                                               
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                   
GluHisTrpSerHisAspProPheGlu                                               
15                                                                        
(2) INFORMATION FOR SEQ ID NO:3:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 1224 base pairs                                               
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: cDNA                                                  
(ix) FEATURE:                                                             
(A) NAME/KEY: CDS                                                         
(B) LOCATION: 1..1224                                                     
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                   
ATGACCAGCAAGGGTCCCGAGGAGGAGCACCCATCGGTGACGCTCTTC48                        
MetThrSerLysGlyProGluGluGluHisProSerValThrLeuPhe                          
151015                                                                    
CGCCAGTACCTGCGTATCCGCACTGTCCAGCCCAAGCCTGACTATGGA96                        
ArgGlnTyrLeuArgIleArgThrValGlnProLysProAspTyrGly                          
202530                                                                    
GCTGCTGTGGCTTTCTTTGAGGAGACAGCCCGCCAGCTGGGCCTGGGC144                       
AlaAlaValAlaPhePheGluGluThrAlaArgGlnLeuGlyLeuGly                          
354045                                                                    
TGTCAGAAAGTAGAGGTGGCACCTGGCTATGTGGTGACCGTGTTGACC192                       
CysGlnLysValGluValAlaProGlyTyrValValThrValLeuThr                          
505560                                                                    
TGGCCAGGCACCAACCCTACACTCTCCTCCATCTTGCTCAACTCCCAC240                       
TrpProGlyThrAsnProThrLeuSerSerIleLeuLeuAsnSerHis                          
65707580                                                                  
ACGGATGTGGTGCCTGTCTTCAAGGAACATTGGAGTCACGACCCCTTT288                       
ThrAspValValProValPheLysGluHisTrpSerHisAspProPhe                          
859095                                                                    
GAGGCCTTCAAGGATTCTGAGGGCTACATCTATGCCAGGGGTGCCCAG336                       
GluAlaPheLysAspSerGluGlyTyrIleTyrAlaArgGlyAlaGln                          
100105110                                                                 
GACATGAAGTGCGTCAGCATCCAGTACCTGGAAGCTGTGAGGAGGCTG384                       
AspMetLysCysValSerIleGlnTyrLeuGluAlaValArgArgLeu                          
115120125                                                                 
AAGGTGGAGGGCCACCGGTTCCCCAGAACCATCCACATGACCTTTGTG432                       
LysValGluGlyHisArgPheProArgThrIleHisMetThrPheVal                          
130135140                                                                 
CCTGATGAGGAGGTTGGGGGTCACCAAGGCATGGAGCTGTTCGTGCAG480                       
ProAspGluGluValGlyGlyHisGlnGlyMetGluLeuPheValGln                          
145150155160                                                              
CGGCCTGAGTTCCACGCCCTGAGGGCAGGCTTTGCCCTGGATGAGGGC528                       
ArgProGluPheHisAlaLeuArgAlaGlyPheAlaLeuAspGluGly                          
165170175                                                                 
ATAGCCAATCCCACTGATGCCTTCACTGTCTTTTATAGTGAGCGGAGT576                       
IleAlaAsnProThrAspAlaPheThrValPheTyrSerGluArgSer                          
180185190                                                                 
CCCTGGTGGGTGCGGGTTACCAGCACTGGGAGGCCAGGCCATGCCTCA624                       
ProTrpTrpValArgValThrSerThrGlyArgProGlyHisAlaSer                          
195200205                                                                 
CGCTTCATGGAGGACACAGCAGCAGAGAAGCTGCACAAGGTTGTAAAC672                       
ArgPheMetGluAspThrAlaAlaGluLysLeuHisLysValValAsn                          
210215220                                                                 
TCCATCCTGGCATTCCGGGAGAAGGAATGGCAGAGGCTGCAGTCAAAC720                       
SerIleLeuAlaPheArgGluLysGluTrpGlnArgLeuGlnSerAsn                          
225230235240                                                              
CCCCACCTGAAAGAGGGGTCCGTGACCTCCGTGAACCTGACTAAGCTA768                       
ProHisLeuLysGluGlySerValThrSerValAsnLeuThrLysLeu                          
245250255                                                                 
GAGGGTGGCGTGGCCTATAACGTGATACCTGCCACCATGAGCGCCAGC816                       
GluGlyGlyValAlaTyrAsnValIleProAlaThrMetSerAlaSer                          
260265270                                                                 
TTTGACTTCCGTGTGGCACCGGATGTGGACTTCAAGGCTTTTGAGGAG864                       
PheAspPheArgValAlaProAspValAspPheLysAlaPheGluGlu                          
275280285                                                                 
CAGCTGCAGAGCTGGTGCCAGGCAGCTGGCGAGGGGGTCACCCTAGAG912                       
GlnLeuGlnSerTrpCysGlnAlaAlaGlyGluGlyValThrLeuGlu                          
290295300                                                                 
TTTGCTCAGAAGTGGATGCACCCCCAAGTGACACCTACTGATGACTCA960                       
PheAlaGlnLysTrpMetHisProGlnValThrProThrAspAspSer                          
305310315320                                                              
AACCCTTGGTGGGCAGCTTTTAGCCGGGTCTGCAAGGATATGAACCTC1008                      
AsnProTrpTrpAlaAlaPheSerArgValCysLysAspMetAsnLeu                          
325330335                                                                 
ACTCTGGAGCCTGAGATCATGCCTGCTGCCACTGACAACCGCTATATC1056                      
ThrLeuGluProGluIleMetProAlaAlaThrAspAsnArgTyrIle                          
340345350                                                                 
CGCGCGGTGGGGGTCCCAGCTCTAGGCTTCTCACCCATGAACCGCACA1104                      
ArgAlaValGlyValProAlaLeuGlyPheSerProMetAsnArgThr                          
355360365                                                                 
CCTGTGCTGCTGCACGACCACGATGAACGGCTGCATGAGGCTGTGTTC1152                      
ProValLeuLeuHisAspHisAspGluArgLeuHisGluAlaValPhe                          
370375380                                                                 
CTCCGTGGGGTGGACATATATACACGCCTGCTGCCTGCCCTTGCCAGT1200                      
LeuArgGlyValAspIleTyrThrArgLeuLeuProAlaLeuAlaSer                          
385390395400                                                              
GTGCCTGCCCTGCCCAGTGACAGC1224                                              
ValProAlaLeuProSerAspSer                                                  
405                                                                       
(2) INFORMATION FOR SEQ ID NO:4:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 408 amino acids                                               
(B) TYPE: amino acid                                                      
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: protein                                               
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                   
MetThrSerLysGlyProGluGluGluHisProSerValThrLeuPhe                          
151015                                                                    
ArgGlnTyrLeuArgIleArgThrValGlnProLysProAspTyrGly                          
202530                                                                    
AlaAlaValAlaPhePheGluGluThrAlaArgGlnLeuGlyLeuGly                          
354045                                                                    
CysGlnLysValGluValAlaProGlyTyrValValThrValLeuThr                          
505560                                                                    
TrpProGlyThrAsnProThrLeuSerSerIleLeuLeuAsnSerHis                          
65707580                                                                  
ThrAspValValProValPheLysGluHisTrpSerHisAspProPhe                          
859095                                                                    
GluAlaPheLysAspSerGluGlyTyrIleTyrAlaArgGlyAlaGln                          
100105110                                                                 
AspMetLysCysValSerIleGlnTyrLeuGluAlaValArgArgLeu                          
115120125                                                                 
LysValGluGlyHisArgPheProArgThrIleHisMetThrPheVal                          
130135140                                                                 
ProAspGluGluValGlyGlyHisGlnGlyMetGluLeuPheValGln                          
145150155160                                                              
ArgProGluPheHisAlaLeuArgAlaGlyPheAlaLeuAspGluGly                          
165170175                                                                 
IleAlaAsnProThrAspAlaPheThrValPheTyrSerGluArgSer                          
180185190                                                                 
ProTrpTrpValArgValThrSerThrGlyArgProGlyHisAlaSer                          
195200205                                                                 
ArgPheMetGluAspThrAlaAlaGluLysLeuHisLysValValAsn                          
210215220                                                                 
SerIleLeuAlaPheArgGluLysGluTrpGlnArgLeuGlnSerAsn                          
225230235240                                                              
ProHisLeuLysGluGlySerValThrSerValAsnLeuThrLysLeu                          
245250255                                                                 
GluGlyGlyValAlaTyrAsnValIleProAlaThrMetSerAlaSer                          
260265270                                                                 
PheAspPheArgValAlaProAspValAspPheLysAlaPheGluGlu                          
275280285                                                                 
GlnLeuGlnSerTrpCysGlnAlaAlaGlyGluGlyValThrLeuGlu                          
290295300                                                                 
PheAlaGlnLysTrpMetHisProGlnValThrProThrAspAspSer                          
305310315320                                                              
AsnProTrpTrpAlaAlaPheSerArgValCysLysAspMetAsnLeu                          
325330335                                                                 
ThrLeuGluProGluIleMetProAlaAlaThrAspAsnArgTyrIle                          
340345350                                                                 
ArgAlaValGlyValProAlaLeuGlyPheSerProMetAsnArgThr                          
355360365                                                                 
ProValLeuLeuHisAspHisAspGluArgLeuHisGluAlaValPhe                          
370375380                                                                 
LeuArgGlyValAspIleTyrThrArgLeuLeuProAlaLeuAlaSer                          
385390395400                                                              
ValProAlaLeuProSerAspSer                                                  
405                                                                       
(2) INFORMATION FOR SEQ ID NO:5:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 12 amino acids                                                
(B) TYPE: amino acid                                                      
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: peptide                                               
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                   
GluTrpGlnArgLeuGlnSerAsnProHisLeuLys                                      
1510                                                                      
(2) INFORMATION FOR SEQ ID NO:6:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 11 amino acids                                                
(B) TYPE: amino acid                                                      
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: peptide                                               
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                   
LeuGluGlyGlyValAlaTyrAsnValIlePro                                         
1510                                                                      
(2) INFORMATION FOR SEQ ID NO:7:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 6 amino acids                                                 
(B) TYPE: amino acid                                                      
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: peptide                                               
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                   
GluGlyTyrIleTyrAla                                                        
15                                                                        
(2) INFORMATION FOR SEQ ID NO:8:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 17 base pairs                                                 
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: cDNA                                                  
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                   
GAGGGGTATATATATGC17                                                       
(2) INFORMATION FOR SEQ ID NO:9:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 5 amino acids                                                 
(B) TYPE: amino acid                                                      
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: peptide                                               
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                   
AsnProHisLeuLys                                                           
15                                                                        
(2) INFORMATION FOR SEQ ID NO:10:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 14 base pairs                                                 
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: cDNA                                                  
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                  
TTGAGGTGGGGGTT14                                                          
(2) INFORMATION FOR SEQ ID NO:11:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 14 base pairs                                                 
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: cDNA                                                  
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                  
TTTAAGTGGGGGTT14                                                          
__________________________________________________________________________

Claims (2)

We claim:
1. A DNA which encodes a protein having the following physicochemial properties:
(1) Molecular weight
45,000±5,000;
Isoelectric point
pI=5.7±0.5;
(3) Partial amino acid sequence
Possessing a partial amino acid sequence of Asp-Ser-Glu-Gly-Tyr-Ile-Tyr-Ala-Arg-Gly-Ala-Gln-Asp-Met-Lys (SEQ ID NO:1) or Glu-His-Trp-Ser-His-Asp-Pro-Phe-Glu (SEQ ID NO:2);
(4) Solubility in solvent
Soluble in water, physiological saline and phosphate buffer;
(5) Biological activity
Exhibiting metastasis-inhibitory activity on RPMI 4788 cell (FERM BP -2429), an established cell line derived from human colon cancer; and
(6) Stability
Inactivated in water at pH 7.2 and 100° C. for 30 minutes
Stable in water at pH 7.2 and 4° C. for one month.
(7) Acute toxicity
Exhibiting an LD50 of 50 mg/kg or higher in mouse when intravenously administered to the mouse.
2. The DNA of claim 1, which has the following base sequence as shown in Chemical formula 2(SEQ ID NO:3). ##STR3##
US08/555,860 1992-09-28 1995-11-13 DNA encoding protein possessing metastasis-inhibitory activity Expired - Fee Related US5585474A (en)

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6548066B1 (en) 1997-05-12 2003-04-15 Aphton Corporation Immunogenic compositions to the CCK-B/gastrin receptor and methods for the treatment of tumors
US20030086941A1 (en) * 1997-05-12 2003-05-08 Dov Michaeli Immunogenic compositions to the CCK-B/gastrin receptor and methods for the treatment of tumors
US20040001842A1 (en) * 1997-05-12 2004-01-01 Dov Michaeli Immunogenic compositions to the CCK-B/gastrin receptor and methods for the treatment of tumors
US8388966B2 (en) 2001-03-23 2013-03-05 Cancer Advances, Inc. Combination treatment of pancreatic cancer
US8343930B2 (en) 2001-05-04 2013-01-01 Cancer Advances, Inc. Combination therapy for the treatment of tumors
US20070249005A1 (en) * 2003-03-28 2007-10-25 Stephen Grimes Gastrin hormone immunoassays
US7964371B2 (en) 2003-03-28 2011-06-21 Cancer Advances, Inc. Gastrin hormone immunoassays
US8158128B2 (en) 2004-09-22 2012-04-17 Cancer Advances, Inc. Monoclonal antibodies to progastrin
US8808695B2 (en) 2004-09-22 2014-08-19 Cancer Advances, Inc. Monoclonal antibodies to progastrin
US11583576B2 (en) 2017-06-15 2023-02-21 Cancer Advances Inc. Compositions and methods for inducing humoral and cellular immunities against tumors and cancer
US12076383B2 (en) 2017-06-15 2024-09-03 Cancer Advances Inc. Compositions and methods for inducing humoral and cellular immunities against tumors and cancer
US12150978B2 (en) 2017-06-15 2024-11-26 Cancer Advances Inc. Compositions and methods for preventing tumors and cancer

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EP0594311A1 (en) 1994-04-27
DE69331368T2 (en) 2002-08-08
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KR940007056A (en) 1994-04-26
US5827691A (en) 1998-10-27

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